PROJECT SUMMARY Critical knowledge gaps in our understanding of stem cells and their epigenetic control mechanisms are roadblocks to clinical translation of stem cell-related technologies. Advances in our understanding of these processes will have substantial impact on human health by catalyzing innovative disease treatments and development of regenerative medicine therapies. Our long-term goal is to accelerate the development of innovative stem cell-related treatments by determining the epigenetic mechanisms of stem cell functions, including cellular plasticity. The focused objective here, which is the next step towards that goal, is to elucidate the roles of histone variant H3.3 in regulating plasticity in stem cells and to modulate its functions. Our central hypothesis is that H3.3 acts as a novel, targetable epigenetic switch: in a nave stem cell state H3.3 confers plasticity, but it can toggle to promote differentiation in response to specific signals. The rationale for the proposed studies is that once H3.3 epigenetic switch functions are defined and models of switch modulation are created, our work will have high translational impact by catalyzing new approaches to treat infertility, birth defects, and cancer. Our multidisciplinary team of stem cell researchers will test our hypothesis via three Specific Aims. The first aim focuses on mechanisms of H3.3 regulation of induced plasticity during cellular reprogramming to make induced pluripotent stem cells (IPSC). The second aim will determine mechanisms of maintained plasticity by H3.3 in ESCs. The third aim interrogates and modulates H3.3 function in differentiation and differentiated cells. In each aim H3.3 and switch components will be modulated genetically via coordinated, synergistic interventions including novel H3.3 knockout mice and next generation CRISPR-Cas9 gene editing in human cells. The proposed research is significant because the knowledge gained here and the creation of model systems of H3.3 switch modulation will be a vertical advance, laying the foundation for transformative therapies for human diseases. Our proposal is innovative in key ways: 1) the hypothesis that H3.3 can function as a controllable epigenetic switch brings fresh ways of thinking to the cell and developmental biology and epigenetic fields, opening the door to therapeutically directing cellular plasticity and differentiation; 2) a focus on novel specific H3.3 functions including specific target genes, cofactors such as PRC2 and MYC, and 3D chromatin mechanisms; and 3) the new combination of powerful tools (e.g. H3.3 KO mice and cellular reprogramming) and innovative approaches such as single cell transcriptomics and gene editing of human cells. Overall, important advances in the knowledge and future treatment of stem cell-related diseases are expected. In addition, a deeper fundamental understanding of the mechanisms of epigenetic switches and cellular plasticity are anticipated.